Nucleic acid molecules from plants coding enzymes which participate in the starch synthesis

ABSTRACT

Nucleic acid molecules encoding enzymes which participate in the starch synthesis in plants are described. These enzymes are a novel isotype of starch synthase.  
     Furthermore, the invention relates to vectors and hose cells which were transformed with the described nucleic acid molecules, in particular to transformed plant cells and to plants which may be regenerated therefrom and which exhibit an increased or reduced activity of the described proteins.

[0001] The present invention relates to nucleic acid molecules encodingan enzyme participating in the starch synthesis in plants. This enzymeis a novel isotype of the starch synthase. Furthermore, this inventionrelates to vectors, bacteria as well as to plant cells transformed withthe described nucleic acid molecules and to plants which may be obtainedfrom these plant cells by means of regeneration.

[0002] Furthermore, methods for the production of transgenic plants aredescribed, which due to the introduction of DNA molecules encoding astarch synthase synthesize a starch modified in its properties.

[0003] With respect to the increasing significance which has recentlybeen ascribed to vegetal substances as regenerative sources of rawmaterials, one of the objects of biotechnological research is trying toadapt vegetal raw materials to the demands of the processing industry.In order to enable the use of regenerative raw materials in as manyareas as possible, it is furthermore important to obtain a large varietyof substances. Apart from oils, fats and proteins, polysaccharidesconstitute the essential regenerative raw materials derived from plants.Apart from cellulose, starch maintains an important position among thepolysaccharides, being one of the most significant storage substances inhigher plants. Among those, maize is one of the most interesting plantsas it is the most important cultivated plant for the production ofstarch.

[0004] The polysaccharide starch is a polymer made up of chemicallyhomogeneous basic components, namely the glucose molecules. However, itconstitutes a highly complex mixture of various types of molecules whichdiffer from each other in their degree of polymerization and in thedegree of branching of the glucose chains. Therefore, starch is not ahomogeneous raw material. One differentiates particularly betweenamylose-starch, a basically non-branched polymer made up ofα-1,4-glycosidically branched glucose molecules, and amylopectin-starchwhich in turn is a complex mixture of various branched glucose chains.The branching results from additional α-1,6-glycosidic interlinkings. Inplants used typically for the production of starch, such as maize orpotato, the synthesized starch consists of approximately 25%amylose-starch and of about 75% amylopectin-starch.

[0005] In order to enable as wide a use of starch as possible, it seemsto be desirable that plants be provided which are capable ofsynthesizing modified starch which is particularly suitable for varioususes. One possibility to provide such plants—apart from breedingmethods—is the specific genetic modification of the starch metabolism ofstarch-producing plants by means of recombinant DNA techniques. However,a prerequisite therefore is to identify and to characterize the enzymesinvolved in the starch synthesis and/or the starch modification as wellas to isolate the respective DNA molecules encoding these enzymes.

[0006] The biochemical pathways which lead to the synthesis of starchare basically known. The starch synthesis in plant cells takes place inthe plastids. In photosynthetically active tissues these are thechloroplasts, in photosynthetically inactive, starch-storing tissues theamyloplasts.

[0007] The most important enzymes involved in starch synthesis arestarch synthases as well as branching enzymes. In the case of starchsynthases various isotypes are described which all catalyze apolymerization reaction by transferring a glucosyl residue ofADP-glucose to α-1,4-glucans. Branching enzymes catalyze theintroduction of α-1,6 branchings into linear α-1,4-glucans.

[0008] Starch synthases may be divided up in two groups: thegranule-bound starch synthases (GBSS) and the soluble starch synthases(SSS). This distinction is not always evident since some starchsynthases are granule-bound as well as soluble (Denyer et al., Plant J.4 (1993), 191-198; Mu et al., Plant J. 6 (1994), 151-159). Within theseclassifications, various isotypes are described for various species ofplants. These isotypes differ from each other in their dependency onprimer molecules (so-called “primer dependent” (type II) and “primerindependent” (type I) starch synthases).

[0009] So far only in the case of the isotype GBSS I its exact functionduring starch synthesis has been successfully determined. Plants inwhich this enzyme activity has been strongly or completely reduced,synthesize starch free of amylose (a so-called “waxy” starch) (Shure etal., Cell 35 (1983), 225-233; Visser et al., Mol. Gen. Genet. 225(1991), 289-296; WO 92/11376); therefore this enzyme has been assigned adecisive role in synthesizing amylose-starch. This phenomenon is alsoobserved in the cells of the green alga Chlamydomonas reinhardtii(Delrue et al., J. Bacteriol. 174 (1992), 3612-3620). In the case ofChlamydomonas it was furthermore demonstrated that GBSS I is not onlyinvolved in the synthesis of amylose but also has a certain influence onamylopectin synthesis. In mutants which do not show any GBSS I activitya certain fraction of the normally synthesized amylopectin, exhibitinglong chain glucans, is missing.

[0010] The functions of the other isotypes of the granule-bound starchsynthases, particularly GBSS II, and of the soluble starch synthases areso far not clear. It is assumed that soluble starch synthases, togetherwith branching enzymes, are involved in the synthesis of amylopectin(see e.g. Ponstein et al., Plant Physiol. 92 (1990), 234-241) and thatthey play an important role in the regulation of starch synthesis rate.

[0011] In the case of maize, two isotypes of the starch granule-boundstarch synthase as well as two or respectively three isotypes of thesoluble starch synthases were identified (Hawker et al., Arch. Biochem.Biophys. 160 (1974), 530-551; Pollock and Preiss, Arch. Biochem.Biophys. 204 (1980), 578-588; MacDonald and Preiss, Plant Physiol. 78(1985), 849-852; Mu et al., Plant J. 6 (1994), 151-159).

[0012] A cDNA encoding GBSS I from maize and a genomic DNA have alreadybeen described (Shure et al., Cell 35 (1983), 225-233; Kloesgen et al.,Mol. Gen. Genet. 203 (1986), 237-244). Moreover, a so-called “expressedsequence tag” (EST) has been described (Shen et al., 1994, GenBank No.:T14684); the amino acid sequence derived therefrom has a strongsimilarity to the amino acid sequence derived from the GBSS II from pea(Dry et al., Plant J. 2 (1992), 193-202) and potato (Edwards et al.,Plant J. 8 (1995), 283-294). Nucleic acid sequences encoding furtherstarch synthase-isotypes from maize are yet unknown. cDNA sequencesencoding starch synthases other than GBSS I have so far only beendescribed for pea (Dry et al., Plant J. 2 (1992), 193-202), rice (Babaet al., Plant Physiol. 103 (1993), 565-573) and potatoes (Edwards etal., Plant J. 8 (1995), 283-294).

[0013] Soluble starch synthases have been identified in several otherplant species apart from maize. Soluble starch synthases have forexample been isolated in homogeneous form from pea (Denyer and Smith,Planta 186 (1992), 609-617) and potatoes (Edwards et al., Plant J. 8(1995), 283-294). In these cases it was found that the isotype of thesoluble starch synthase identified as SSS II is identical with thegranule-bound starch synthase GBSS II (Denyer et al., Plant J. 4 (1993),191-198; Edwards et al., Plant J. 8 (1995), 283-294). In the case ofsome other plant species the existence of several SSS-isotypes wasdescribed by means of chromatographic methods, as for example in thecase of barley (Tyynelä and Schulman, Physiologia Plantarum 89 (1993)835-841; Kreis, Planta 148 (1980), 412-416) and wheat (Rijven, PlantPhysiol. 81 (1986), 448-453). However, DNA sequences encoding theseproteins have so far not been described.

[0014] In order to provide further possibilities for modifying anydesired starch-storing plant in such a way that it will synthesize amodified starch, respective DNA sequences encoding further isotypes ofstarch synthases have to be identified.

[0015] Therefore, the technical problem underlying the present inventionis to provide nucleic acid molecules encoding enzymes involved in starchbiosynthesis and by means of which genetically modified plants may beproduced that show an elevated or reduced activity of those enzymes,thereby prompting a modification in the chemical and/or physicalproperties of the starch synthesized in these plants.

[0016] This problem has been solved by the provision of the embodimentsdescribed in the claims.

[0017] Therefore, the present invention relates to nucleic acidmolecules encoding proteins with the biological activity of a starchsynthase, wherein such molecules preferably encode proteins whichcomprise the amino acid sequence depicted under Seq ID No. 2. Theinvention particularly relates to nucleic acid molecules which compriseall or part of the nucleotide sequence mentioned under Seq ID No. 1,preferably molecules, which comprise the coding region indicated in SeqID No. 1 or, as the case may be, corresponding ribonucleotide sequences.Nucleic acid molecules that encode a starch synthase and the sequence ofwhich differs from the nucleotide sequences of the above-mentionedmolecules due to the degeneracy of the genetic code are also thesubject-matter of the invention.

[0018] The present invention further relates to nucleic acid moleculesencoding a starch synthase and hybridizing to one of the above-mentionedmolecules.

[0019] The invention also relates to nucleic acid molecules showing asequence which is complementary to the whole or to a part of thesequence of the above-mentioned nucleic acid molecules.

[0020] The nucleic acid molecules of the invention may be DNA as well asRNA molecules. Corresponding DNA molecules are for instance genomic orcDNA molecules. The RNA molecules may for example be MRNA or antisenseRNA molecules.

[0021] Within the framework of the present invention the term“hybridization” signifies hybridization under conventional hybridizingconditions, preferably under stringent conditions, as described forexample in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2ndEdition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.). Nucleic acid molecules hybridizing to the nucleic acid moleculesof the invention may basically be derived from any desired type oforganism (i.e. prokaryotes or eukaryotes, in particular from bacteria,fungi, algae, plants or animal organisms) comprising such molecules.They are preferably derived from monocotyledonous or dicotyledonousplants, in particular from useful plants, and particularly preferredfrom starch-storing plants, in particular from maize.

[0022] Nucleic acid molecules hybridizing to the molecules of theinvention may for example be isolated from genomic or cDNA libraries ofvarious organisms.

[0023] The identification and isolation of such nucleic acid moleculesfrom plants and other organisms may take place by using the molecules ofthe invention or parts of these molecules or, as the case may be, thereverse complements of these molecules, e.g. by hybridization accordingto standard methods (see e.g. Sambrook et al., 1989, Molecular Cloning,A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.).

[0024] As a probe for hybridization e.g. nucleic acid molecules may beused which exactly or basically contain the nucleotide sequenceindicated under Seq ID No. 1 or parts thereof. The fragments used ashybridization probe may also be synthetic fragments which were producedby means of the conventional synthesizing methods and the sequence ofwhich is basically identical with that of a nucleic acid molecule of theinvention. After identifying and isolating the genes hybridizing to thenucleic acid sequences of the invention, the sequence has to bedetermined and the properties of the proteins encoded by this sequencehave to be analyzed.

[0025] The molecules hybridizing to the nucleic acid molecules of theinvention also comprise fragments, derivatives and allelic variants ofthe above-described nucleic acid molecules which encode a proteinaccording to the invention. In this context, fragments are defined asparts of the nucleic acid molecules, which are long enough in order toencode one of the described proteins. In this context, the termderivative means that the sequences of these molecules differ from thesequences of the above-mentioned nucleic acid molecules at one or morepositions and that they exhibit a high degree of homology to thesesequences. In this regard, homology means a sequence identity of atleast 40%, in particular an identity of at least 60%, preferably of morethan 80% and still more preferably a sequence identity of more than 90%.The deviations occurring when comparing with the above-described nucleicacid molecules might have been caused by deletion, substitution,insertion or recombination.

[0026] Moreover, homology means that functional and/or structuralequivalence exists between the respective nucleic acid molecules or theproteins they encode. The nucleic acid molecules, which are homologousto the above-described molecules and represent derivatives of thesemolecules, are generally variations of these molecules, that constitutemodifications which exert the same biological function. These variationsmay be naturally occurring variations, for example sequences derivedfrom other organisms, or mutations, wherein these mutations may haveoccurred naturally or they may have been introduced by means of aspecific mutagenesis. Moreover the variations may be syntheticallyproduced sequences. The allelic variants may be naturally occurring aswell as synthetically produced variants or variants produced byrecombinant DNA techniques.

[0027] The proteins encoded by the various variants of the nucleic acidmolecules according to the invention exhibit certain commoncharacteristics. Enzyme activity, molecular weight, immunologicreactivity, conformation etc. may belong to these characteristics aswell as physical properties such as the mobility in gel electrophoresis,chromatographic characteristics, sedimentation coefficients, solubility,spectroscopic properties, stability; pH-optimum, temperature-optimumetc.

[0028] Significant characteristics of a starch synthase are: i) itslocalization within the stroma of the plastids of plant cells; ii) itscapability of synthesizing linear α-1,4-linked polyglucans usingADP-glucose as substrate. This activity can be determined as shown inDenyer and Smith (Plants 186 (1992), 606-617) or as described in theexamples.

[0029] The nucleic acid molecules of the invention may in principle bederived from any desired organism expressing the described proteins,preferably from plants and in particular from starch-synthesizing orstarch-storing plants. They may be monocotyledonous as well asdicotyledonous plants. Cereals (such as barley, rye, oats, wheat etc.),maize, rice, pea, cassava or potato etc. are particularly preferred.

[0030] The proteins encoded by the nucleic acid molecules of theinvention represent a so far not identified and not characterizedisotype of a plant starch synthase. These proteins exhibit the enzymaticactivity of a starch synthase as well as certain regions of homology tostarch synthases from plants known so far; however, they may not beunambiguously classified as any of the isotypes known so far. Inparticular, the proteins encoded by the nucleic acid molecules of theinvention have the property that they lead to a blue staining of thebacterial colonies after their introduction into an E.coli mutant, inwhich all glg genes are deleted and which expresses a mutated,deregulated ADP glucose-pyrophosphorylase, cultivation of this mutant ona glucose-containing medium and staining with iodine vapor.

[0031] Another subject matter of the invention are oligonucleotideswhich hybridize specifically with a nucleic acid molecule of theinvention. Such oligonucleotides preferably have a length of at least10, in particular of at least 15 and particularly preferred of at least50 nucleotides. These oligonucleotides are characterized in that theyspecifically hybridize with the nucleic acid molecules of the invention,i.e. that they do not or only to a very limited extent hybridize withnucleic acid sequences encoding other proteins, in particular otherstarch synthases. The oligonucleotides of the invention may for examplebe used as primers for a PCR reaction. They may also be components ofantisense constructs or of DNA molecules encoding suitable ribozymes.

[0032] Furthermore, the invention relates to vectors, especiallyplasmids, cosmids, viruses, bacteriophages and other vectors common ingenetic engineering, which contain the above-mentioned nucleic acidmolecules of the invention.

[0033] In a preferred embodiment the nucleic acid molecules contained inthe vectors are linked to regulatory elements that ensure thetranscription and synthesis of a translatable RNA in prokaryotic andeukaryotic cells.

[0034] The expression of the nucleic acid molecules of the invention inprokaryotic cells, e.g. in Escherichia coli, is interesting insofar asthis enables a more precise characterization of the enzymatic activitiesof the enzymes encoding these molecules. In particular, it is possibleto characterize the product being synthesized by the respective enzymesin the absence of other enzymes which are involved in the starchsynthesis of the plant cell. This makes it possible to draw conclusionsabout the function, which the respective protein exerts during thestarch synthesis within the plant cell.

[0035] Moreover, it is possible to introduce various mutations into thenucleic acid molecules of the invention by means of conventionalmolecular-biological techniques (see e.g. Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), whereby the synthesis ofproteins with possibly modified biological properties can be achieved.By means of this it is on the one hand possible to produce deletionmutants, in which nucleic acid molecules are produced by continuingdeletions at the 5′- or the 3′-end of the encoding DNA-sequence. Thesenucleic acid molecules may lead to the synthesis of correspondinglyshortened proteins. Such deletions at the 5′-end of the nucleotidesequence make it possible, for example, to identify amino acid sequenceswhich are responsible for the translocation of the enzyme in theplastids (transit peptides). This allows for the specific production ofenzymes which due to the removal of the respective sequences are nolonger located in the plastids but within the cytosole, or which due tothe addition of other signal sequences are located in othercompartments.

[0036] On the other hand point mutations might also be introduced atpositions where a modification of the amino acid sequence influences,for example, the enzyme activity or the regulation of the enzyme. Inthis way e.g. mutants with a modified K_(m)-value may be produced, ormutants which are no longer subject to the regulation mechanisms byallosteric regulation or covalent modification usually occurring incells.

[0037] Furthermore, mutants may be produced exhibiting a modifiedsubstrate or product specificity such as mutants that useADP-glucose-6-phosphate instead of ADP-glucose as substrate. Moreover,mutants with a modified activity-temperature-profile may be produced.

[0038] For the genetic manipulation in prokaryotic cells the nucleicacid molecules of the invention or parts of these molecules may beintegrated into plasmids which allow for a mutagenesis or a sequencemodification by recombination of DNA sequences. By means of standardmethods (cf. Sambrook et al., 1989, Molecular Cloning: A laboratorymanual, 2nd edition, Cold Spring Harbor Laboratory Press, N.Y., USA)base exchanges may be carried out or natural or synthetic sequences maybe added. In order to connect the DNA fragments, adapters or linkers maybe attached to the fragments. Moreover, use can be made of manipulationswhich offer suitable restriction sites or which remove superfluous DNAor restriction sites. Wherever use is made of inserts, deletions orsubstitutions, in vitro mutagenesis, “primer repair”, restriction orligation may be used. For analyzing use is usually made of a sequenceanalysis, a restriction analysis or furtherbiochemico-molecularbiological methods.

[0039] In a further embodiment the invention relates to host cells, inparticular prokaryotic or eukaryotic cells, which have been transformedand genetically modified by an above-mentioned nucleic acid molecule ofthe invention or by a vector of the invention, as well as cells derivedfrom such transformed cells and containing a nucleic acid molecule or avector of the invention. This is preferably a bacterial cell or a plantcell.

[0040] Furthermore, the proteins encoded by the nucleic acid moleculesof the invention or biologically active fragments thereof are thesubject-matter of the invention as well as methods for their productionwherein a host cell of the invention is cultivated under conditions thatallow for the synthesis of the protein and wherein the protein is thenisolated from the cultivated cells and/or the culture medium.

[0041] By the provision of the nucleic acid molecules of the inventionit is now possible—by means of recombinant DNA techniques—tospecifically interfere with the starch metabolism of plants in a way sofar impossible by means of breeding. Thereby, the starch metabolism maybe modified in such a way that a modified starch is synthesized whiche.g. is modified, compared to the starch synthesized in wildtype plants,with respect to its physico-chemical properties, especially theamylose/amylopectin ratio, the degree of branching, the average chainlength, the phosphate content, the pastification behavior, the size ofthe starch granules and/or the shape of the starch granules. There isthe possibility of increasing the yield of genetically modified plantsby increasing the activity of the proteins described in the invention,e.g. by overexpressing the respective nucleic acid molecules or bymaking mutants available which are no longer subject to cell-specificregulation schemes and/or different temperature-dependencies withrespect to their activity. The economic significance of the chance tointerfere with the starch synthesis of maize alone is obvious since 80%of all starch produced annually in the world is produced from starch.

[0042] Therefore, it is possible to express the nucleic acid moleculesof the invention in plant cells in order to increase the activity of therespective starch synthase. Furthermore, the nucleic acid molecules ofthe invention may be modified by means of methods known to the skilledperson, in order to produce starch synthases according to the inventionwhich are no longer subject to the cell-specific regulation mechanismsor show modified temperature-dependencies or substrate or productspecificities.

[0043] In expressing the nucleic acid molecules of the invention inplants the synthesized proteins may in principle be located in anydesired compartment within the plant cell. In order to locate it withina specific compartment, the sequence ensuring the localization in theplastids must be deleted and the remaining coding region optionally hasto be linked to DNA sequences which ensure localization in therespective compartment. Such sequences are known (see e.g. Braun et al.,EMBO J. 11 (1992), 3219-3227; Wolter et al., Proc. Natl. Acad. Sci. USA85 (1988), 846-850; Sonnewald et al., Plant J. 1 (1991), 95-106).

[0044] Thus, the present invention also relates to transgenic plantcells transformed and genetically modified with a nucleic acid moleculeof the invention, as well as it relates to transgenic plant cells whichare derived from cells transformed in such a way. Such cells contain anucleic acid molecule of the invention, wherein this is preferablylinked to regulatory DNA elements, which ensure the transcription inplant cells, especially with a promoter. The transgenic plant cells ofthe invention differ from naturally occurring plant cells in that theycontain integrated into their genome a DNA molecule of the inventionwhich either does not naturally occur in such cells at all or only in adifferent genomic environment, i.e. at a different position in thegenome. Plant cells which may contain naturally in their genome a DNAmolecule of the invention differ from the plant cells of the inventionin that the latter exhibit more gene copies of the nucleic acidmolecules of the invention than would naturally occur in the respectivenaturally occurring plant cells and in that these additional copies areintegrated at different genetic positions. The above-mentioned featuresmay for example be determined by means of Southern Blot analysis ofgenomic DNA. By means of methods known to the skilled person thetransgenic plant cells can be regenerated to whole plants. Thus, theplants obtained by regenerating the transgenic plant cells of theinvention are also the subject-matter of the present invention. Afurther subject-matter of the invention are plants which contain theabove-described transgenic plant calls. The transgenic plants may inprinciple be plants of any desired species, i.e. they may bemonocotyledonous as well as dicotyledonous plants. They are preferablyuseful plants, in particular starch-synthesizing or starch-storingplants such as cereals (rye, barley, oats, wheat etc.), rice, maize,peas, cassava or potatoes.

[0045] The invention also relates to propagation material of the plantsof the invention, e.g. fruits, seeds, tubers, rootstocks, seedlings,cuttings etc.

[0046] Due to the expression or, as the case may be, additionalexpression of a nucleic acid molecule of the invention, the transgenicplant cells and plants described in the invention synthesize a starchwhich compared to starch synthesized in wildtype plants is modified forexample in its physico-chemical properties, in particular in theamylose/amylopectin ratio, the degree of branching, the averagechain-length, the phosphate content, the pastification behavior, thesize of the starch granules and/or the shape of the starch granules.Compared with wildtype-starch, such starch may be modified in particularwith respect to its viscosity and/or the gel formation properties of theglues of this starch.

[0047] Thus, also the starch obtainable from transgenic plant cells andplants according to the invention is the subject-matter of the presentinvention.

[0048] By means of the nucleic acid molecules of the invention it isfurthermore possible to produce plant cells and plants in which theactivity a protein of the invention is reduced. This also leads to thesynthesis of a starch with modified chemical and/or physical propertieswhen compared to the starch from wildtype plant cells.

[0049] Thus, transgenic plant cells, in which the activity of a proteinaccording to the invention is reduced when compared to non-transformedplants, are a further subject-matter of the invention.

[0050] The production of plant cells with a reduced activity of aprotein of the invention may for example be achieved by the expressionof a corresponding antisense-RNA, of a sense-RNA for achieving acosupression effect or the expression of a correspondingly constructedribozyme, which specifically cleaves transcripts encoding one of theproteins of the invention, using the nucleic acid molecules of theinvention.

[0051] In order to reduce the activity of a protein of the inventionantisense-RNA is preferably expressed in plant cells.

[0052] In order to express an antisense-RNA, on the one hand DNAmolecules can be used which comprise the complete sequence encoding aprotein of the invention, including possibly existing flanking sequencesas well as DNA molecules, which only comprise parts of the codingregion. These parts have to be long enough in order to prompt anantisense-effect within the cells. Basically, sequences with a minimumlength of 15 bp, preferably with a length of 100-500 bp and for anefficient antisense-inhibition, in particular sequences with a length ofmore than 500 bp may be used. Generally DNA-molecules are used which areshorter than 5000 bp, preferably sequences with a length of less than2500 bp.

[0053] Use may also be made of DNA sequences which are highlyhomologous, but not completely identical to the sequences of the DNAmolecules of the invention. The minimal homology should be more thanabout 65%. Preferably, use should be made of sequences with homologiesbetween 95 and 100%.

[0054] The cells of the invention differ from naturally occurring cellsin that they contain a heterologous recombinant DNA molecule encoding anantisense RNA, a ribozyme or a cosuppression RNA. Due to the expressionof this heterologous recombinant DNA molecule the synthesis of a proteinof the invention in the cells is reduced and thereby also thecorresponding activity.

[0055] In this context “heterologous” DNA means that the DNA introducedinto the cells is a DNA not naturally occurring in the cells in thisform. On the one hand, it may be DNA which does naturally not at alloccur in these transformed cells or DNA which, even if it does occur inthese cells, is integrated at other genetic positions as exogenous DNAand is therefore situated within another genetic environment.

[0056] The transgenic plant cells of the invention can be regenerated towhole plants by means of methods known to the skilled person. Thus,plants containing the transgenic plant cells of the invention are alsothe subject-matter of the present invention. These plants may inprincipal be plants of any desired plant species, i.e. monocotyledonousas well as dicotyledonous plants. Preferably, they are useful plants,i.e. plants cultivated by man for the nourishment of humans or animalsor for technical purposes. Particularly preferred, they arestarch-synthesizing or starch-storing plants such as cereals (rye,barley, oats, wheat, etc.), rice, maize, pea, cassava or potato. Theinvention also relates to propagation material of the plants of theinvention such as fruits, seeds, tubers, rootstocks, seedlings, cuttingsetc.

[0057] Due to the reduction of the activity of a protein of theinvention, the transgenic plant cells and plants of the inventionsynthesize a starch which is modified, compared to the starchsynthesized in wildtype plants, for example, in its physico-chemicalproperties, in particular in the amylose/amylopectin ratio, the degreeof branching, the average chain-length, the phosphate-content, thepastification behavior, the size of the starch granules and/or the shapeof the starch granules. This starch may for example exhibit modifiedviscosities and/or gel formation properties of its glues when comparedto starch derived from wildtype plants. Thus, starch obtainable from theabove-mentioned transgenic plant cells and plants also is thesubject-matter of the present invention.

[0058] The starches of the invention may be modified according totechniques known to the skilled person; in unmodified as well as inmodified form they are suitable for the use in foodstuffs and for theuse in non-foodstuffs.

[0059] Basically, the possibilities of uses of the starch can besubdivided into two major fields. One field comprises the hydrolysisproducts of starch, essentially glucose and glucans components obtainedby enzymatic or chemical processes. They can be used as startingmaterial for further chemical modifications and processes, such asfermentation. In this context, it might be of importance that thehydrolysis process can be carried out simply and inexpensively.Currently, it is carried out substantially enzymatically usingamyloglucosidase. It is thinkable that costs might be reduced by usinglower amounts of enzymes for hydrolysis due to changes in the starchstructure, e.g. increasing the surface of the grain, improveddigestibility due to less branching or a steric structure, which limitsthe accessibility for the used enzymes.

[0060] The other field in which the starch is used because of itspolymer structure as so-called native starch, can be subdivided into twofurther areas:

[0061] 1. Use in foodstuffs

[0062] Starch is a classic additive for various foodstuffs, in which itessentially serves the purpose of binding aqueous additives and/orcauses an increased viscosity or an increased gel formation. Importantcharacteristic properties are flowing and sorption behavior, swellingand pastification temperature, viscosity and thickening performance,solubility of the starch, transparency and paste structure, heat, shearand acid resistance, tendency to retrogradation, capability of filmformation, resistance to freezing/thawing, digestibility as well as thecapability of complex formation with e.g. inorganic or organic ions.

[0063] A preferred area of application of native starch is the field ofbakery-goods and pasta.

[0064] 2. Use in non-foodstuffs

[0065] The other major field of application is the use of starch as anadjuvant in various production processes or as an additive in technicalproducts. The major fields of application for the use of starch as anadjuvant are, first of all, the paper and cardboard industry. In thisfield, the starch is mainly used for retention (holding back solids),for sizing filler and fine particles, as solidifying substance and fordehydration. In addition, the advantageous properties of starch withregard to stiffness, hardness, sound, grip, gloss, smoothness, tearstrength as well as the surfaces are utilized.

[0066] 2.1 Paper and cardboard industry

[0067] Within the paper production process, a differentiation can bemade between four fields of application, namely surface, coating, massand spraying.

[0068] The requirements on starch with regard to surface treatment areessentially a high degree of brightness, corresponding viscosity, highviscosity stability, good film formation as well as low formation ofdust. When used in coating the solid content, a corresponding viscosity,a high capability to bind as well as a high pigment affinity play animportant role. As an additive to the mass rapid, uniform, loss-freedispersion, high mechanical stability and complete retention in thepaper pulp are of importance. When using the starch in spraying,corresponding content of solids, high viscosity as well as highcapability to bind are also significant.

[0069] 2.2 Adhesive industry

[0070] A major field of application is, for instance, in the adhesiveindustry, where the fields of application are subdivided into fourareas: the use as pure starch glue, the use in starch glues preparedwith special chemicals, the use of starch as an additive to syntheticresins and polymer dispersions as well as the use of starches asextenders for synthetic adhesives. 90% of all starch-based adhesives areused in the production of corrugated board, paper sacks and bags,composite materials for paper and aluminum, boxes and wetting glue forenvelopes, stamps, etc.

[0071] 2.3 Textile and textile care industry

[0072] Another possible use as adjuvant and additive is in theproduction of textiles and textile care products. Within the textileindustry, a differentiation can be made between the following fourfields of application: the use of starch as a sizing agent, i.e. as anadjuvant for smoothing and strengthening the burring behavior for theprotection against tensile forces active in weaving as well as for theincrease of wear resistance during weaving, as an agent for textileimprovement mainly after quality-deteriorating pretreatments, such asbleaching, dying, etc., as thickener in the production of dye pastes forthe prevention of dye diffusion and as an additive for warping agentsfor sewing yarns.

[0073] 2.4 Building industry

[0074] The fourth area of application of starch is its use as anadditive in building materials. One example is the production of gypsumplaster boards, in which the starch mixed in the thin plaster pastifieswith the water, diffuses at the surface of the gypsum board and thusbinds the cardboard to the board. Other fields of application areadmixing it to plaster and mineral fibers. In ready-mixed concrete,starch may be used for the deceleration of the sizing process.

[0075] 2.5 Ground stabilization

[0076] Furthermore, the starch is advantageous for the production ofmeans for ground stabilization used for the temporary protection ofground particles against water in artificial earth shifting. Accordingto state-of-the-art knowledge, combination products consisting of starchand polymer emulsions can be considered to have the same erosion- andencrustation-reducing effect as the products used so far; however, theyare considerably less expensive.

[0077] 2.6 Use of starch in plant protectives and fertilizers

[0078] Another field of application is the use of starch in plantprotectives for the modification of the specific properties of thesepreparations. For instance, starches are used for improving the wettingof plant protectives and fertilizers, for the dosed release of theactive ingredients, for the conversion of liquid, volatile and/orodorous active ingredients into microcristalline, stable, deformablesubstances, for mixing incompatible compositions and for theprolongation of the duration of the effect due to a reduceddisintegration.

[0079] 2.7 Drugs, medicine and cosmetics industry

[0080] Starch may also be used in the fields of drugs, medicine and inthe cosmetics industry. In the pharmaceutical industry, the starch maybe used as a binder for tablets or for the dilution of the binder incapsules. Furthermore, starch is suitable as disintegrant for tabletssince, upon swallowing, it absorbs fluid and after a short time itswells so much that the active ingredient is released. For qualitativereasons, medicinal flowance and dusting powders are further fields ofapplication. In the field of cosmetics, the starch may for example beused as a carrier of powder additives, such as scents and salicylicacid. A relatively extensive field of application for the starch istoothpaste.

[0081] 2.8 Starch as an additive in coal and briquettes

[0082] The use of starch as an additive in coal and briquettes is alsothinkable. By adding starch, coal can be quantitatively agglomeratedand/or briquetted in high quality, thus preventing prematuredisintegration of the briquettes. Barbecue coal contains between 4 and6% added starch, calorated coal between 0.1 and 0.5%. Furthermore, thestarch is suitable as a binding agent since adding it to coal andbriquette can considerably reduce the emission of toxic substances.

[0083] 2.9 Processing of ore and coal slurry

[0084] Furthermore, the starch may be used as a flocculant in theprocessing of ore and coal slurry.

[0085] 2.10 Starch as an additive in casting

[0086] Another field of application is the use as an additive to processmaterials in casting. For various casting processes cores produced fromsands mixed with binding agents are needed. Nowadays, the most commonlyused binding agent is bentonite mixed with modified starches, mostlyswelling starches.

[0087] The purpose of adding starch is increased flow resistance as wellas improved binding strength. Moreover, swelling starches may fulfillmore prerequisites for the production process, such as dispersability incold water, rehydratisability, good mixability in sand and highcapability of binding water.

[0088] 2.11 Use of starch in rubber industry

[0089] In the rubber industry starch may be used for improving thetechnical and optical quality. Reasons for this are improved surfacegloss, grip and appearance. For this purpose, the starch is dispersed onthe sticky rubberized surfaces of rubber substances before the coldvulcanization. It may also be used for improving the printability ofrubber.

[0090] 2.12 Production of leather substitutes

[0091] Another field of application for the modified starch is theproduction of leather substitutes.

[0092] 2.13 Starch in synthetic polymers

[0093] In the plastics market the following fields of application areemerging: the integration of products derived from starch into theprocessing process (starch is only a filler, there is no direct bondbetween synthetic polymer and starch) or, alternatively, the integrationof products derived from starch into the production of polymers (starchand polymer form a stable bond).

[0094] The use of the starch as a pure filler cannot compete with othersubstances such as talcum. This situation is different when the specificstarch properties become effective and the property profile of the endproducts is thus clearly changed. One example is the use of starchproducts in the processing of thermoplastic materials, such aspolyethylene. Thereby, starch and the synthetic polymer are combined ina ratio of 1:1 by means of coexpression to form a ‘master batch’, fromwhich various products are produced by means of common techniques usinggranulated polyethylene. The integration of starch in polyethylene filmsmay cause an increased substance permeability in hollow bodies, improvedwater vapor permeability, improved antistatic behavior, improvedanti-block behavior as well as improved printability with aqueous dyes.

[0095] Another possibility is the use of the starch in polyurethanefoams. Due to the adaptation of starch derivatives as well as due to theoptimization of processing techniques, it is possible to specificallycontrol the reaction between synthetic polymers and the starch's hydroxygroups. The results are polyurethane films having the following propertyprofiles due to the use of starch: a reduced coefficient of thermalexpansion, decreased shrinking behavior, improved pressure/tensionbehavior, increased water vapor permeability without a change in wateracceptance, reduced flammability and cracking density, no drop off ofcombustible parts, no halides and reduced aging. Disadvantages thatpresently still exist are reduced pressure and impact strength.

[0096] Product development of film is not the only option. Also solidplastics products, such as pots, plates and bowls can be produced bymeans of a starch content of more than 50%. Furthermore, thestarch/polymer mixtures offer the advantage that they are much easierbiodegradable.

[0097] Furthermore, due to their extreme capability to bind water,starch graft polymers have gained utmost importance. These are productshaving a backbone of starch and a side lattice of a synthetic monomergrafted on according to the principle of radical chain mechanism. Thestarch graft polymers available nowadays are characterized by animproved binding and retaining capability of up to 1000 g water per gstarch at a high viscosity. These super absorbers are used mainly in thehygiene field, e.g. in products such as diapers and sheets, as well asin the agricultural sector, e.g. in seed pellets.

[0098] What is decisive for the use of the new starch modified byrecombinant DNA techniques are, on the one hand, structure, watercontent, protein content, lipid content, fiber content, ashes/phosphatecontent, amylose/amylopectin ratio, distribution of the relative molarmass, degree of branching, granule size and shape as well ascrystallization, and on the other hand, the properties resulting in thefollowing features: flow and sorption behavior, pastificationtemperature, viscosity, thickening performance, solubility, pastestructure, transparency, heat, shear and acid resistance, tendency toretrogradation, capability of gel formation, resistance tofreezing/thawing, capability of complex formation, iodine binding filmformation, adhesive strength, enzyme stability, digestibility andreactivity.

[0099] The production of modified starch by genetically operating with atransgenic plant may modify the properties of the starch obtained fromthe plant in such a way as to render further modifications by means ofchemical or physical methods superfluous. On the other hand, thestarches modified by means of recombinant DNA techniques might besubjected to further chemical modification, which will result in furtherimprovement of the quality for certain of the above-described fields ofapplication. These chemical modifications are principally known to theperson skilled in the art. These are particularly modifications by meansof

[0100] heat treatment

[0101] acid treatment

[0102] oxidation and

[0103] esterification

[0104] leading to the formation of phosphate, nitrate, sulfate,xanthate, acetate and citrate starches. Other organic acids may also beused for the esterification:

[0105] formation of starch ethers

[0106] starch alkyl ether, O-allyl ether, hydroxylalkyl ether,O-carboxylmethyl ether, N-containing starch ethers, P-containing starchethers and S-containing starch ethers.

[0107] formation of branched starches

[0108] formation of starch graft polymers.

[0109] In order to express the nucleic acid molecules of the inventionin sense- or antisense-orientation in plant cells, these are linked toregulatory DNA elements which ensure the transcription in plant cells.Such regulatory DNA elements are particularly promoters. Basically anypromoter which is active in plant cells may be used for the expression.

[0110] The promoter may be selected in such a way that the expressiontakes place constitutively or only in a certain tissue, at a certainpoint of time of the plant development or at a point of time determinedby external factors. With respect to the plant the promoter may behomologous or heterologous. Suitable promoters for a constitutiveexpression are, e.g. the 35S RNA promoter of the Cauliflower MosaicVirus and the ubiquitin promoter from maize. For a tuber-specificexpression in potatoes the patatin gene promoter B33 (Rocha-Sosa et al.,EMBO J. 8 (1989), 23-29) may be used or a promoter can be used whichensures expression only in photosynthetically active tissues, e.g. theST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987),7943-7947; Stockhaus et al., EMBO J. 8 (1989), 2445-2451). For anendosperm-specific expression the HMG promoter from wheat, the USPpromoter, the phaseolin promoter or promoters from zein genes from maizeare suitable.

[0111] Furthermore, a termination sequence may be present, which servesto correctly end the transcription and to add a poly-A-tail to thetranscript which is believed to stabilize the transcripts. Such elementsare described in the literature (cf. Gielen et al., EMBO J. 8 (1989),23-29) and can be exchanged as desired.

[0112] The present invention provides nucleic acid molecules encoding anovel isotype of a starch synthase identified in maize. This allows forthe identification of the function of this isotype in the starchbiosynthesis as well as for the production of genetically modifiedplants in which the activity of this enzyme is modified. This enablesthe synthesis of starch with a modified structure and therefore withmodified physico-chemical properties in the plants manipulated in such away. In principal, the nucleic acid molecules of the invention may alsobe used in order to produce plants in which the activity of the starchsynthase of the invention is elevated or reduced and in which at thesame time the activities of other enzymes involved in the starchbiosynthesis are modified. Thereby, all kinds of combinations andpermutations are thinkable. By modifying the activity of one or moreisotypes of the starch synthases in plants, a synthesis of a starchmodified in its structure is brought about. By increasing the activityof one or more isotypes of the starch synthases in the cells of thestarch-storing tissue of transformed plants, such as in the endosperm ofmaize or wheat or in the potato tuber, increased yields may be theresult. For example, nucleic acid molecules encoding a protein of theinvention, or corresponding antisense-constructs may be introduced intoplant cells in which the synthesis of endogenous GBSS I-, SSS- or GBSSII-proteins is already inhibited due to an antisense-effect or amutation, or in which the synthesis of the branching enzyme is inhibited(as described e.g. WO92/14827 or in connection with the ae mutant(Shannon and Garwood, 1984, in Whistler, BeMiller and Paschall, Starch:Chemistry and Technology, Academic Press, London, 2^(nd) edition:25-86)).

[0113] If the inhibition of the synthesis of several starch synthases intransformed plants is to be achieved, DNA molecules can be used fortransformation, which at the same time contain several regions inantisense-orientation controlled by a suitable promoter and encoding thecorresponding starch synthases. In such constructs, each sequence mayalternatively be controlled by its own promoter or else the sequencesmay be transcribed as a fusion from a common promoter. The lastalternative will generally be preferred as in this case the synthesis ofthe respective proteins should be inhibited to approximately the sameextent.

[0114] Furthermore it is possible to construct DNA molecules in whichapart from DNA sequences encoding starch synthases other DNA sequencesare present encoding other proteins involved in the starch synthesis ormodification and coupled to a suitable promoter in antisenseorientation. Again, the sequences may be connected up in series and betranscribed from a common promoter. For the length of the individualcoding regions used in such a construct the above-mentioned factsconcerning the production of antisense-construct are also true. There isno upper limit for the number of antisense fragments transcribed from apromoter in such a DNA molecule. The resulting transcript, however,should not be longer than 10 kb, preferably 5 kb.

[0115] Coding regions which are located in antisense-orientation behinda suitable promoter in such DNA molecules in combination with othercoding regions, may be derived from DNA sequences encoding the followingproteins: granule-bound starch synthases (GBSS I and II), other solublestarch synthases (SSS I and II), branching enzymes, debranching enzymes,disproportionizing enzymes and starch phosphorylases. This enumerationmerely serves as an example. The use of other DNA sequences within theframework of such a combination is also thinkable.

[0116] By means of such constructs it is possible to inhibit thesynthesis of several enzymes at the same time within the plant cellstransformed with these molecules.

[0117] Furthermore, the constructs may be introduced into classicalmutants which are defective for one or more genes of the starchbiosynthesis (Shannon and Garwood, 1984, in Whistler, BeMiller andPaschall, Starch: Chemistry and Technology, Academic Press, London,2^(nd) edition: 25-86). These defects may be related to the followingproteins: granule-bound (GBSS I and II) and soluble starch synthases(SSS I and II), branching enzymes (BE I and II), debranching enzymes(R-enzymes), disproportionizing enzymes and starch phosphorylases. Thisenumeration merely serves as an example.

[0118] By means of such strategy it is furthermore possible to inhibitthe synthesis of several enzymes at the same time within the plant cellstransformed with these molecules. In order to prepare the introductionof foreign genes into higher plants a high number of cloning vectors areat disposal, containing a replication signal for E.coli and a markergene for the selection of transformed bacterial cells. Examples for suchvectors are pBR322, pUC series, M13mp series, pACYC184 etc. The desiredsequence may be integrated into the vector at a suitable restrictionsite. The obtained plasmid is used for the transformation of E.colicells. Transformed E.coli cells are cultivated in a suitable medium andsubsequently harvested and lysed. The plasmid is recovered. As ananalyzing method for the characterization of the obtained plasmid DNAuse is generally made of restriction analysis, gel electrophoresis andother biochemico-molecularbiological methods. After each manipulationthe plasmid DNA may be cleaved and the obtained DNA fragments may belinked to other DNA sequences. Each plasmid DNA may be cloned into thesame or in other plasmids.

[0119] In order to introduce DNA into a plant host cell a wide range oftechniques are at disposal. These techniques comprise the transformationof plant cells with T-DNA by using Agrobacterium tumefaciens orAgrobacterium rhizogenes as transformation medium, the fusion ofprotoplasts, the injection and the electroporation of DNA, theintroduction of DNA by means of the biolistic method as well as furtherpossibilities.

[0120] In the case of injection and electroporation of DNA into plantcells, there are no special demands made to the plasmids used. Simpleplasmids such as pUC derivatives may be used. However, in case thatwhole plants are to be regenerated from cells transformed in such a way,a selectable marker gene should be present.

[0121] Depending on the method of introducing desired genes into theplant cell, further DNA sequences may be necessary. If the Ti- orRi-plasmid is used e.g. for the transformation of the plant cell, atleast the right border, more frequently, however, the right and leftborder of the Ti- and Ri-plasmid T-DNA should be connected to theforeign gene to be introduced as a flanking region.

[0122] If Agrobacteria are used for the transformation, the DNA which isto be integrated must be cloned into special plasmids, namely eitherinto an intermediate vector or into a binary vector. Due to sequenceshomologous to the sequences within the T-DNA, the intermediate vectorsmay be integrated into the Ti- or Ri-plasmid of the Agrobacterium due tohomologous recombination. This also contains the vir-region necessaryfor the transfer of the T-DNA. Intermediate vectors cannot replicate inAgrobacteria. By means of a helper plasmid the intermediate vector maybe transferred to Agrobacterium tumefaciens (conjugation). Binaryvectors may replicate in E.coli as well as in Agrobacteria. They containa selectable marker gene as well as a linker or polylinker which isframed by the right and the left T-DNA border region. They may betransformed directly into the Agrobacteria (Holsters et al. Mol. Gen.Genet. 163 (1978), 181-187). The Agrobacterium acting as host cellshould contain a plasmid carrying a vir-region. The vir-region isnecessary for the transfer of the T-DNA into the plant cell. AdditionalT-DNA may be present. The Agrobacterium transformed in such a way isused for the transformation of plant cells.

[0123] The use of T-DNA for the transformation of plant cells wasinvestigated intensely and described sufficiently in EP 120 516;Hoekema, In: The Binary Plant Vector System Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant.Sci., 4, 1-46 and An et al. EMBO J. 4 (1985), 277-287.

[0124] For transferring the DNA into the plant cells, plant explants maysuitably be co-cultivated with Agrobacterium tumefaciens orAgrobacterium rhizogenes. From the infected plant material (e.g. piecesof leaves, stem segments, roots, but also protoplasts orsuspension-cultivated plant cells) whole plants may then be regeneratedin a suitable medium which may contain antibiotics or biozides for theselection of transformed cells. The plants obtained in such a way maythen be examined as to whether the introduced DNA is present or not.Other possibilities in order to introduce foreign DNA by using thebiolistic method or by transforming protoplasts are known to the skilledperson (cf. e.g. Willmitzer, L., 1993 Transgenic plants. In:Biotechnology, A Multi-Volume Comprehensive Treatise (H. J. Rehm, G.Reed, A. Pühler, P. Stadler, editors), Vol. 2, 627-659, VCH Weinheim-NewYork-Basel-Cambridge).

[0125] Whereas the transformation of dicotyledonous plants viaTi-plasmid vector systems by means of Agrobacterium tumefaciens is wellestablished, more recent studies indicate that also monocotyledonousplants may be suitable for the transformation by means of vectors basedon Agrobacterium (Chan et al., Plant Mol. Biol. 22 (1993), 491-506; Hieiet al., Plant J. 6 (1994), 271-282).

[0126] Alternative Systems for the transformation of monocotyledonousplants are the transformation by means of a biolistic approach,protoplast transformation, the electroporation of partiallypermeabilized cells, the introduction of DNA by means of glass fibers.

[0127] There are various references in the relevant literature dealingspecifically with the transformation of maize (cf. e.g. WO95/06128, EP 0513 849; EP 0 465′ 875). In EP 292 435 a method is described by means ofwhich fertile plants may be obtained starting from mucousless, friablegranulous maize callus. In this context it was furthermore observed byShillito et al. (Bio/Technology 7 (1989), 581) that for regeneratingfertile plants it is necessary to start from callus-suspension culturesfrom which a culture of dividing protoplasts can be produced which iscapable to regenerate to plants. After an in vitro cultivation period of7 to 8 months Shillito et al. obtain plants with viable descendantswhich, however, exhibited abnormalities in morphology andreproductivity.

[0128] Prioli and Söndahl (Bio/Technology 7 (1989), 589) have describedhow to regenerate and to obtain fertile plants from maize protoplasts ofthe Cateto maize inbreed line Cat 100-1. The authors assume that theregeneration of protoplast to fertile plants depends on a number ofvarious factors such as the genotype, the physiological state of thedonor-cell and the cultivation conditions.

[0129] Once the introduced DNA has been integrated in the genome of theplant cell, it usually continues to be stable there and also remainswithin the descendants of the originally transformed cell. It usuallycontains a selectable marker which confers resistance against biozidesor against an antibiotic such as kanamycin, G 418, bleomycin, hygromycinor phosphinotricine etc. to the transformed plant cells. Theindividually selected marker should therefore allow for a selection oftransformed cells against cells lacking the introduced DNA.

[0130] The transformed cells grow in the usual way within the plant (seealso McCormick et al., Plant Cell Reports 5 (1986), 81-84). Theresulting plants can be cultivated in the usual way and cross-bred withplants having the same transformed genetic heritage or another geneticheritage. The resulting hybrid individuals have the correspondingphenotypic properties.

[0131] Two or more generations should be grown in order to ensurewhether the phenotypic feature is kept stably and whether it istransferred. Furthermore, seeds should be harvested in order to ensurethat the corresponding phenotype or other properties will remain.

[0132] The examples illustrate the invention.

[0133] Abbreviations used bp base pair GBSS granule bound starchsynthase IPTG isopropyl β-D-thiogalacto pyranoside SS starch synthaseSSS soluble starch synthase

[0134] Media and solutions used in the examples: 20 x SSC: 175.3 g NaCl88.2 g sodium citrate ad 1000 ml with ddH₂O ph 7.0 with 10 N NaOH YT 8 gBacto-Yeast extract 5 g Bacto-Tryptone 5 g NaCl ad 1000 ml with ddH₂O

[0135] Protoplast isolation medium (100 ml) Cellulase Onozuka R S (MeijiSeika, Japan) 800 mg Pectolyase Y 23 40 mg KNO₃ 200 mg KH₂PO₄ 136 mgK₂HPO₄ 47 mg CaCl₂ 2H₂O 147 mg MgSO₄ 7H₂O 250 mg Bovine serum albumine(BSA) 20 mg Glucose 4000 mg Fructose 4000 mg Sucrose 1000 mg pH 5.8Osmolarity 660 mosm.

[0136] Protoplast washing solution 1: like protoplast isolationsolution, but without cellulase, pectolyase and BSA.

[0137] Transformation buffers: a) Glucose 0.5 M MES 0.1% MgCl₂ 6H₂O 25mM pH 5.8 adjust to 600 mosm. b) PEG 6000-solution Glucose 0.5 M MgCl₂6H₂O 100 mM Hepes  20 mM pH 6.5

[0138] PEG 6000 is added to the buffer described in b) immediately priorto the use of the solution (40% w/v PEG). The solution is filtered witha 0.45 μm sterile filter.

[0139] W5 solution CaCl₂ 125 mM NaCl 150 mM KCl  5 mM Glucose  50 mM

[0140] Protoplast culture medium (indicated in mg/l) KNO₃ 3000 (NH₄)₂SO₄500 MgSO₄ 7H₂O 350 KH₂PO₄ 400 CaCl₂ 2H₂O 300

[0141] Fe-EDTA and trace elements as in the Murashige-Skoog medium(Physiol. Plant, 15 (1962), 473). m-inosite 100 Thiamine HCl 1.0Nicotine acid amide 0.5 Pyridoxine HCl 0.5 Glycine 2.0 Glucuronic acid750 Galacturonic acid 750 Galactose 500 Maltose 500 Glucose 36.000Fructose 36.000 Sucrose 30.000 Asparagine 500 Glutamine 100 Proline 300Caseinhydrolysate 500

[0142] 2,4 dichlorophenoxy acetic acid (2,4-D) 0.5 pH 5.8 Osmolarity 600mosm.

[0143] In the example the following methods were used:

[0144] 1. Cloning

[0145] For cloning in E.coli the vector pBluescript II SK (Stratagene)was used.

[0146] 2. Bacterial strains

[0147] For the Bluescript vector and for the pUSP constructs use wasmade of the E.coli strain DH5α (Bethesda Research Laboratories,Gaithersburgh, USA). For in vivo excision the E.coli strain XL1-Blue wasused.

[0148] 3. Transformation of maize

[0149] (a) Production of protoplasts of the cell line DSM 6009

[0150] Protoplast isolation

[0151] 2-4 days, preferably 3 days after the last change of medium in aprotoplast suspension culture the liquid medium is pumped off and theremaining cells are washed in 50 ml protoplast washing solution 1 andsucked dry once more. 10 ml protoplast isolation medium are added to 2 gof harvested cell mass. The resuspended cells and cell aggregates areincubated at 27±2° C. for 4 to 6 hours in the darkness, while shaking itslightly (at 30 to 40 rpm).

[0152] Protoplast purification

[0153] As soon as the release of at least 1 million protoplasts/ml hastaken place (microscopic inspection), the suspension is sifted through astainless steel or nylon sieve with a mesh size of 200 or 45 μm. Thecombination of a 100 μm and a 60 μm sieve allows for separating the cellaggregates just as well. The protoplast-containing filtrate is examinedmicroscopically. It usually contains 98-99% protoplasts. The rest areundigested single cells. Protoplast preparations with such a degree ofpurity are used for transformation experiments without additionalgradient centrifugation. The protoplasts are sedimented by means ofcentrifugation (100 UpM in the swing-out rotor (100×g, 3 minutes)). Thesupernatant is abandoned and the protoplasts are resuspended in washingsolution 1. The centrifugation is repeated and the protoplasts aresubsequently resuspended in the transformation buffer.

[0154] (b) Protoplast transformation

[0155] The protoplasts resuspended in the transformation biffer arefilled in 10 ml portions into 50 ml polyallomer tubes at a titer of0.5-1×10⁶ protoplasts/ml. The DNA used for transformation is dissolvedin Tris-EDTA (TE) buffer solution. 20 μg plasmid DNA is added to each mlprotoplast suspension. A plasmid which provides for resistance tophosphinotricine is used as vector (cf. e.g. EP 0 513 849). After theaddition of DNA the protoplast suspension is carefully shaken in orderto homogeneously distribute the DNA in the solution. Immediatelyafterwards 5 ml PEG solution is added in drops.

[0156] By carefully shaking the tubes the PEG solution is distributedhomogeneously. Afterwards further 5 ml of PEG solution are added and thehomogenous mixing is repeated. The protoplasts remain in the PEGsolution for 20 minutes at ±2° C. Afterwards the protoplasts aresedimented by centrifuging for 3 minutes (100 g; 1000 Upm). Thesupernatant is abandoned. The protoplasts are washed in 20 ml W5solution by careful shaking and are again subjected to centrifugation.Then they are resuspended in 20 ml protoplast culture medium,centrifuged anew and again resuspended in culture medium. The titer isadjusted to 6−8×10⁵ protoplasts and the protoplasts are cultivated in 3ml portions in Petri dishes (Ø 60 mm, height 15 mm). The Petri dishesare sealed with a parafilm and stored in darkness at 25±2° C.

[0157] (c) Protoplast culture

[0158] During the first 2-3 weeks after the protoplast isolation andtransformation the protoplasts are cultivated without adding freshmedium. As soon as the cells regenerated from the protoplasts havedeveloped into cell aggregates with more than 20 to 50 cells, 1 ml offresh protoplast culture medium, containing sucrose as an osmotic (90g/l), is added.

[0159] (d) Selection of transformed maize cells and plant regeneration

[0160] 3-10 days after adding fresh medium the cell aggregates developedfrom the protoplasts may be plated on Agar media with 100 mg/lL-phosphinothricine. N6-medium with the vitamins of the protoplastculture medium, 90 g/l sucrose and 1.0 mg/l 2,4D is as suitable as ananalogous medium such as a medium with the macro- and micro-nutritivesalts of the MS medium (Murashige and Skoog (1962), see above).

[0161] The calli developed from stably transformed protoplasts may growfurther on the selective medium. After 3 to 5 weeks, preferably 4 weeksthe transgenic calli may be transferred to fresh selection medium whichalso contains 100 mg/l L-phosphinothricine which, however, does nolonger contain auxine. Within 3 to 5 weeks approximately 50% of thetransgenic maize calli which had integrated theL-phosphinothricine-acetyl-transferase gene into their genome, start todifferentiate into plants on this medium in the presence ofL-phosphinothricine.

[0162] (e) Growing of transgenic regenerative plants

[0163] The embryogenic transformed maize tissue is cultivated onhormone-free N6-medium (Chu C. C. et al., Sci. Sin. 16 (1975), 659) inthe presence of 5×10⁻⁴ M L-phosphinothricine. On this medium maizeembryos, which express the phosphinothricine-acetyl-transferase gene(PAT gene) in a sufficiently strong manner, develop into plants.Non-transformed embryos or such with only a very weak PAT activity diedown. As soon as the leaves of the in-vitro plants have reached a lengthof 4 to 6 mm, they may be transferred into soil. After washing off theAgar residues at the roots the plants are planted into a mixture ofclay, sand, vermiculite and potting soil with the ratio 3:1:1:1 andadapted to the soil culture at 90-100% of relative atmospheric humidityduring the first 3 days after planting. The growing is carried out in aclimate chamber with a 14 hour light period of approximately 25000 luxat the height of the plant at a day/night temperature of 23±{fraction(1/17)}±1° C. The adapted plants are cultivated at an 65±5% atmospherichumidity.

[0164] 4. Radioactive labeling of DNA fragments

[0165] The radioactive labeling of DNA fragments was carried out bymeans of a DNA-Random Primer Labeling Kits by Boehringer (Germany)according to the manufacturer's instructions.

EXAMPLE 1

[0166] Identification, isolation and characterization of a cDNA encodinga novel isotype of a starch synthase from Zea mays.

[0167] In order to identify a cDNA encoding a novel starch synthase frommaize the strategy of the functional expression in a suitable E.colimutant was pursued. As a storage carbohydrate, E.coli synthesizes onspecific nutrient media (complete medium with 1% glucose) apolysaccharide the structure of which resembles that of amylopectin;however, it exhibits a higher degree of branching (7-10% branchingpoints vis-á-vis 4-5%) and usually a higher molecular weight. The higherdegree of branching also causes a different iodine staining. Iodinegives a brownish staining to glycogen, a purple staining to amylopectinand a blue staining to amylose.

[0168] In E.coli three genes are essentially responsible for glycogensynthesis, namely glgA, glgB and glgC which encode glycogen synthase,the branching enzyme and the ADP glucose pyrophosphorylase. This systemis analogous to that of the starch biosynthesis in plants. If a plantstarch synthase is expressed by wildtype E.coli cells, it is difficultor impossible to determine the influence of the enzyme on the propertiesof the glycogen with the help of the iodine staining since the branchingenzyme introduces branch points in the glucans produced by the starchsynthase in the same way as into the glucans produced by the glycogensynthase.

[0169] Therefore, an E.coli strain was produced which allows for asimple screening after the functional expression of a starch synthase bymeans of iodine staining. For this purpose, the mutant HfrG6MD2(Schwartz, J. Bacteriol. 92 (1966), 1083-1089) in which all glg genesare deleted, was transformed with the plasmid pACAC. This plasmidcontains a DNA fragment encoding the ADP glucose pyrophosphorylase(AGPase) from E.coli under the control of the lac Z promoter. Thefragment had been isolated from the pEcA-15 vector (see e.g.Müller-Röber (1992), dissertation, FU Berlin) as a DraI/HaeII fragmentwith the approximate size of 1.7 kb and after filling in its sticky endsit had been cloned into a pACAC184 vector linearized with HindIII. Thisplasmid mediates the expression of a mutated, deregulated ADP glucosepyrophosphorylase from the E.coli strain LCB 618 which accumulatesconsiderable amounts of glycogen due to the mutation of this enzyme(Preiss and Romeo in Advances in Microbial Physiology, Academic Press,London, Vol. 30, 183-238). This ensures the provision of sufficientamounts of ADP glucose, the substrate of starch synthases, and issupposed to ensure the synthesis of linear α-1,4-glucans in E.coliHfrG6MD2 which may be stained blue by iodine, when simultaneouslyfunctionally expressing starch synthases. Furthermore, the plasmidpACAC, as a derivative of the vector pACYC184 is compatible withplasmids such as the pBluescript SK (−).

[0170] This strain was transformed with a cDNA library contained in thepBluescript SK (−) vector and produced from RNA from leaves of Zea mays,line B73. This was produced by initially converting approximately 10⁶phases of a cDNA library from RNA from leaves of Zea mays, line B37,contained in the Uni-ZAPTMXR vector (Stratagene GmbH, Heidelberg), intophagmides by means of in vivo excision. E.coli XL1-Blue cells wereinfected with these phagmids and 3×10⁵ transformants were plated onsolid selective (ampicilline-containing) nutrient medium. After growththe cells were washed off and plasmid DNA was prepared therefrom. Thetransfer of the plasmid DNA into the bacterial cells was carried outaccording to the method of Hanahan (J. Mol. Biol. 166 (1983), 557-580).Approximately 4×10⁵ transformed E.coli cells were spread on Agar culturemedia having the following composition:

[0171] YT medium with:

[0172] 1.5% Bacto Agar

[0173] 1% glucose

[0174] 10 mg/l chloramphenicol

[0175] 50 mg/l ampicilline

[0176] 1 mM IPTG

[0177] 2 mM diaminopimelinic acid

[0178] After overnight incubation at 37° C. the cells were treated withwith iodine vapor. A blue-stained colony was obtained. From this colony,plasmid DNA was isolated and used for repeated transformation. Theobtained transformants were spread on replica plates. One of the replicaplates was again stained. Blue-staining clones were grown for thepreparation of elevated amounts of plasmid DNA.

[0179] After examining the size of the cDNA fragment the clone pSSZm wasfurther analyzed.

EXAMPLE 2

[0180] Sequence analysis of the cDNA insertion of the plasmid pSSZm

[0181] From an E.coli clone obtained according to example 1, the plasmidpSSZm was isolated and its cDNA insertion was determined by standardroutines using the didesoxynucleotide method (Sanger et al., Proc. Natl.Acad. Sci. USA 74 (1977), 5463-5467). The insertion has a length of 2651bp and constitutes a partial cDNA. The nucleotide sequence is indicatedunder Seq ID No. 1. The corresponding amino acid sequence is shown underSeq ID No. 2.

[0182] A sequence analysis and a sequence comparison with knownsequences showed that the sequence indicated under Seq ID No. 1 is newand comprises a partial coding region which exhibits certain homologiesto starch synthases from various organisms. Moreover, the encodedprotein constitutes a new isotype of starch synthases which may not beunambiguously grouped among the classes described so far. Within theframework of this application the protein encoded by this cDNA insertionor by hybridizing sequences is designated SSZm. By means of this partialcDNA sequence it is possible for a person skilled in the field ofmolecular biology without further ado to isolate the full-length clonescontaining the complete coding region and to determine its sequence. Forthis purpose, e.g. a leaf-specific cDNA expression library from Zeamays, line B73 (Stratagene GmbH, Heidelberg) is screened for full-lengthclones by means of hybridization with a 5′-fragment of the cDNAinsertion of the plasmid pSSZM (200 bp). Another possibility forobtaining the missing 5′-terminal sequences is to make use of the5′-Race method (Stratagene cf. manufacturer).

We claim:
 1. A nucleic acid molecule encoding a protein with thebiological activity of a starch synthase selected from the groupconsisting of: (a) nucleic acid molecules encoding a protein comprisingthe amino acid sequence indicated in Seq ID No. 2; (b) nucleic acidmolecules comprising the nucleotide sequence indicated under Seq ID No.1 or a corresponding ribonucleotide sequence; and (c) nucleic acidmolecules hybridizing to the nucleic acid molecules indicated under (a)or (b) and encoding a protein with starch synthase activity; and therespective complementary strand of such a nucleic acid molecule.
 2. Thenucleic acid molecule of claim 1 which is a DNA molecule.
 3. The DNAmolecule of claim 2 which is a cDNA molecule.
 4. The nucleic acidmolecule of claim 1 which is an RNA molecule.
 5. An oligonucleotidewhich specifically hybridizes with a nucleic acid molecule of any one ofclaims 1 to 4 .
 6. A vector comprising a DNA molecule of any one ofclaim 1 to
 4. 7. The vector of claim 6 wherein the DNA molecule islinked in sense-orientation to regulatory elements which ensure thetranscription and the synthesis of a translatable RNA in prokaryotic oreukaryotic cells.
 8. Host cells which are transformed and geneticallymodified with a nucleic acid molecule of any one of claims 1 to 4 orwith a vector of claim 6 or 7 .
 9. A protein encoded by a nucleic acidmolecule of any one of claims 1 to 4 .
 10. A method for the productionof a protein of claim 9 in which a host cell of claim 8 is cultivatedunder conditions that allow for the synthesis of the protein and inwhich the protein is isolated from the cultivated cells and/or theculture medium.
 11. A transgenic plant cell transformed with a nucleicacid molecule of any one of claims 1 to 4 or with a vector of claim 6 or7 , or derived from such a cell, wherein the nucleic acid moleculeencoding the protein with the biological activity of a starch synthaseis subject to the control of regulatory elements allowing for thetranscription of a translatable MRNA in plant cells.
 12. A plantcomprising plant cells of claim 11 .
 13. The plant of claim 12 which isa useful plant.
 14. The plant of claim 13 which is a starch-storingplant.
 15. The plant of claim 14 which is a maize plant.
 16. Propagationmaterial of a plant of any one of claims 12 to 15 , containing plantcells of claim 11 .
 17. Starch obtainable from a plant of any one ofclaims 12 to 15 .
 18. A transgenic plant cell which is characterized inthat the activity of a protein of claim 9 is reduced in this plant celldue to the expression of a heterologous recombinant DNA molecule which(a) encodes an antisense RNA to the transcripts of genes which encode aprotein of the invention and which occur endogenously in the cells;and/or (b) encodes a ribozyme which specifically cleaves the transcriptsof genes which encode a protein of the invention and which occurendogenously in the cell; and/or (c) an RNA which inhibits the synthesisof the protein of the invention due to a cosuppression effect within thecells.
 19. The plant cell of claim 18 wherein the reduction of theactivity in this cell is achieved by the expression of an antisense-RNAto transcripts of a DNA molecule of claim 1 .
 20. A plant containingplant cells of claim 18 or 19 .
 21. The plant of claim 20 which is auseful plant.
 22. The plant of claim 21 which is a starch-storing plant.23. The plant of claim 22 which is a maize plant.
 24. Propagationmaterial of a plant of any one of claims 20 to 23 containing cells ofclaim 18 or 19 .
 25. Starch obtainable from plants of any one of claims20 to 24 .